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Nanodiamond bioconjugate probes and their collection by electrophoresis
Diamond & Related Materials 17 (2008) 1858–1866
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Diamond & Related Materials j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / d i a m o n d
Nanodiamond bioconjugate probes and their collection by electrophoresis Suzanne Ciftan Hens a,⁎, Garry Cunningham a, Talmage Tyler a, Sergey Moseenkov b, Vladimir Kuznetsov b, Olga Shenderova a a b
International Technology Center, 8100 Brownleigh Drive, Suite 120, Raleigh, NC 27617, United States Boreskov Institute of Catalysis SB RAS, Lavrentiev Ave. 5, Novosibirsk, 630090, Russia
A R T I C L E
I N F O
Article history: Received 29 June 2007 Received in revised form 25 February 2008 Accepted 14 March 2008 Available online 1 April 2008 Keywords Diamond Nanotechnology Surface Chemistry Bioconjugation Electrophoresis Streptavidin
A B S T R A C T The application of detonation nanodiamonds (NDs) as probes for protein capture and electrophoretic collection was investigated. NDs were chemically modified in a series of reactions to produce a ND-NH2 product that had increased chemical homogeneity. The product was characterized by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). FTIR spectra were taken using an IR vacuum cuvette and the samples were dehydrated at different temperatures. The ND-NH2 product was capable of conjugating to N-hydroxysuccinimide derivatives of TAMRA and biotin. We calculated that the number of chemically attached TAMRA molecules on ND-NH2 was ∼ 1 molecule/nm2. The singly conjugated TAMRA-ND (T-ND) and doubly conjugated TAMRA-ND-Biotin (T-ND-B) products formed stable aqueous colloidal suspensions. T-ND and T-ND-B were collected on planar electrodes and silicon field tip arrays using a field of 10 V/cm. The rate of collection for the aminated ND is dependent upon field strength and an exponential decrease in current was observed as a function of time. Streptavidin was captured by the T-ND-B bioconjugate probe and this nanoparticle–protein complex was collected from solution by electrophoresis. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Detonation nanodiamond (DND) particles [1] are an inexpensive complementary material to CVD diamond films and have exceptional properties that are useful for a diverse set of applications, including biomolecular scaffolds, fluorescent probes, and gene delivery vehicles [2,3]. In terms of biomolecular scaffolds, nanodiamonds may physically adsorb proteins and DNA oligos for biomolecular separation and preconcentration [4–6]. As fluorescent probes, NDs may be used as cellular tracer probes due to their low cytotoxicity [7] and their fluorescent properties. And since nanodiamonds are physically strong, DNDs, physisorbed with plasmid DNA, have been used as ballistic delivery vehicles for gene transfection into bacteria [8]. Nanodiamonds fluoresce with different colors, have little photobleaching and blinking characteristics, and are highly luminescent after high energy bombardment that increases their number of nitrogen-vacancy centers [9,10]. Unlike many nanoparticles used as biomolecular probes, the optical transparency of nanodiamonds in the visible wavelength range [11] does not inhibit luminescence of bound fluorophores. In addition, nanodiamonds are compatible as microbiological fluorescent probes since they can be sterilized with high temperature and pressure. ⁎ Corresponding author. Tel.: +1 919 881 0500. E-mail address: [email protected] (S.C. Hens). URL: http://www.itc-inc.org (S.C. Hens). 0925-9635/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.diamond.2008.03.020
Even though the nanodiamond core is physically and chemically strong, its rich surface chemistry is easily modified with a variety of methods. Surface functionalization on diamond includes radical based reactions, producing carboxylic acid, NO2 surface groups [12,13], and fluorine surface groups [14]. Standard wet chemical methods may be used, including the use of fluorinated nanodiamond particles that can be converted to alkyl, amino, and amino acid derivatives [15]. In terms of bioconjugation reactions, nanodiamonds have been derivatized through their direct linkage to reactive surface functional groups to make ND conjugated peptides [16]. And direct conjugation of fluorescent dye and protein has been possible using the free amines from poly-lysine coated nanodiamonds [17]. Nanodiamonds have been used for electrophoretic applications due to their high electrophoretic mobility and controlled surface charges. In fact, electrophoresis of DNDs is a well established area and is used to deposit DNDs as a seed material for CVD growth of diamond films [18,19], and it is used for the co-deposition of a variety of metals during electroplating [20]. While centrifugation is commonly used to collect ND-bound targets from solution, we have found that electrophoresis may also be used for this purpose. Electrophoretic collection of targetbound NDs is practical since the velocity of a ND particle with a high zeta potential of +40 mV will travel a distance of ∼ 2 mm in a short period of time (1 min) using an applied field of only 10 V/cm, as calculated from the particle's electrophoretic mobility [18]. In this work, nanodiamonds are used as target-specific capture probes in order to bind and collect their target, streptavidin, using
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Fig. 1. A schematic representation of the collection of target analytes using nanodiamonds. The T-ND-B nanodiamond probe has biotinylated (B) and TAMRA (T) functionalized to its surface for the capture of the target analyte FITC-streptavidin (S-F) (Step 1). The ND probe mixes and captures streptavidin. Green excitation light may be used to mix and visualize TAMRA emission (Step 2). Under electrophoresis, the captured analyte can be collected from suspension at modest voltages with many types of electrodes, including the field tip array electrode (shown) (Step 3). The captured and collected analyte on the nanodiamond probe may be detected using two excitation wavelengths (Step 4). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
electrophoresis at low electric field strengths. The use of ND in this way is depicted in Fig. 1, where the ND probe is mixed in solution to bind its target and is then collected by electrophoresis for detection by fluorescence emission. To demonstrate this approach, a biotinylated nanodiamond conjugate was synthesized for the specific capture of streptavidin protein. On the same ND surface, a fluorescent dye derivative of rhodamine (carboxytetramethylrhodamine or TAMRA) was attached for detection by fluorescence microscopy. 2. Experimental All chemicals were used without modification. 5-(and-6)carboxytetramethylrhodamine succinimidyl ester (TAMRA-SE, TAMRANHS) and fluorescein isothiocyanate (FITC)-streptavidin were purchased from Invitrogen (South San Francisco, CA). Dry dimethylformamide (DMF) and Sulfo-NHS-SS-Biotin were purchased from Pierce (Rockford, IL). Unlabeled streptavidin, BSA, dimethylsulfoxide (DMSO), dithiolthreitol, potassium carbonate, carbonate buffer, and Tween 20 were purchased from Sigma Aldrich. 2.1. ND-NH2 synthesis The polydispersed nanodiamond material was produced using trinitrotoluene (TNT) and cyclotrimethylenetrinitramine (RDX) explosives by detonation in an ice-cooled chamber. The nanodiamonds have a primary particle size of 4 nm. Ch-St DND and I6 were supplied by New Technologies, Co., Chelyabinsk, Russian Federation. Ch-St was purified using a solution of chromic anhydride in sulfuric acid. The I6 samples were purified from Ch-St by ion-exchange, annealing in air, and fractionation [21]. The ND-NH2 material was produced from polydispersed I6 material containing particle sizes of 20–800 nm with average agglomerate size of 160 nm in a water suspension. Below we provide further details on the ND intermediate products for the synthesis of aminated ND (ND-NH2).
ND-OH(1): An amount of 5 g nanodiamond (I6) was added to a round bottom flask, which was then purged several times using standard Schlenk techniques. To the round bottom flask, 20 mL of degassed anhydrous tetrahydrofuran (THF) and 50 mL of a 2.0 M solution of LiAlH4 in THF were added by cannula. The sample was stirred under a nitrogen atmosphere at room temperature overnight. The reaction was quenched by dropwise addition of 1 M HCl, which solubilized the lithium and aluminum, and then the solution was basified to neutral pH. The product was collected by centrifugation and rinsed several times with water followed by acetone. The product was dried in vacuo at 100 °C for 12 h. X-ray photoelectron spectroscopy (XPS) analysis did not reveal residual lithium or aluminum. ND-OTS(2): To the solid 2 g ND-OH(1) in a round bottom flask an amount of 30 mL of anhydrous pyridine was added by syringe. The solution mixture was sonicated for 5 min. An amount of 1 g of p-toluenesulfonyl chloride or tosyl chloride (OTS) was added and the mixture was stirred at room temperature overnight. The reaction product was collected by centrifugation, rinsed several times with acetone and was dried in vacuo at 100 °C for 12 h. ND-CN(3): 1 g of ND-OTS(2) was placed in round bottom flask with 50 mL of dimethylsulfoxide (DMSO) and sonicated for 10 min. Then 1 g of potassium cyanide was added and the reaction was allowed to stir at 95 °C overnight. The product was collected by centrifugation and rinsed several times with water followed by acetone. The product was dried in vacuo at 100 °C for 12 h. ND-NH2(4): An amount of 500 mg of ND-CN(3) was placed in a round bottom flask, after which the flask was purged several times using standard Schlenk techniques. To the flask, 20 mL of a 2 M solution of LiAlH4 in THF was added and the reaction was allowed to stir at room temperature under a nitrogen atmosphere overnight. The reaction was quenched by dropwise addition of distilled water until hydrogen evolution ceased. The solution then was acidified with 1 M HCl to solubilize lithium and aluminum. The product was collected by centrifugation and washed several times with 2 M NaOH to remove Al
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(OH)3. The product was rinsed with distilled water until the runoff had a neutral pH. The product was dried in vacuo at 100 °C for 12 h. XPS analysis did not reveal residual lithium or aluminum. Zeta potential and particle sizes were measured with a Malvern Zetasizer Nano ZS instrument. The samples were prepared by addition of water to the ND solid, followed by 2 min sonication using a horntype sonicator at 35% power. Samples were measured using Malvern's plastic cuvette. 2.2. ND conjugation reactions The TAMRA-ND and TAMRA-ND-Biotin products were produced by labeling ND-NH2 with their respective succinimidyl ester conjugates (NHS-TAMRA, NHS-Biotin). For the TAMRA-ND-Biotin bioconjugate (T-ND-B), an amount of 30 mg ND-NH2 was added to 5 mL dry DMF and sonicated at 35% power for 4 min before 70 mg K2CO3 was added (pH 8). This was followed by dropwise addition of a solution of 17 mg of sulfo-NHS-SS-Biotin and 1 mg NHS-TAMRA in 1 mL dry DMF. The resulting solution was stirred at room temperature for 90 min and the solid product was collected by centrifugation at 7000 rpm, followed by extensive washing with distilled water. The final concentration of T-ND-B was 30 mg/mL. The same reaction conditions were used to produce the TAMRA-ND product. For the T-ND product, the pellet was colored pink after collection by centrifugation. The presence of an amide bond between TAMRA and ND was tested by hydrolysis. The amine bond was hydrolyzed by the addition of HCl, producing a solution of pH of 1. The solution was centrifuged at 14,000 rpm for 10 min, producing a grey colored pellet and a pink colored supernatant. 2.3. ND surface group characterization It is known, that peaks attributed to OH stretching and bending vibrations of nanodiamond adsorbed water obscure the signatures of other moieties, including NH vibrations that are important for the identification of aminated ND in the present study [22]. Thus in addition to the spectra taken in the air environment, the starting material I6 and the aminated ND samples were analyzed using a vacuum IR cuvette to avoid strong absorbance signals of adsorbed water. Samples for the analysis were prepared by homogenizing of DND in spectral grade KBr in a grinding mill and then pressed into pellets. A fixed quantity of ND sample (25 mg for all samples, except 5 mg of ND-NH2 in air due to its strong absorbance signals) was mixed with KBr powder (800 mg) and pressed with a pressure of up to 150 kg/cm2 into plates 0.5–0.7 mm thick. Tablets were placed in an IR vacuum cell and heated at different temperatures within the range 100–220 °C under vacuum (1 × 10− 2 Torr) for 2–3 h inside an IR cuvette in order to remove traces of water. After this procedure, FTIR spectra were recorded without exposure of the sample to air in order to avoid any influence of atmospheric water. Reference spectra for the empty IR vacuum cuvette (made from KBr plates) were also taken in order to register possible contribution to the sample spectra from the water and CO2 (2370 cm− 1) adsorbed on the external cuvette walls. FTIR analysis was performed using a Shimadzu FTIR-8300 spectrometer. XPS analysis was performed using a Kratos Axis Ultra with a monochromatic Al Kα X-ray source and a silicon wafer as a supporting substrate. The survey and high resolution scans were performed at 160 eV pass energy with 1 eV step and 10 eV pass energy with 0.1 eV step, respectively. The peak position calibration was done against Si 2p3/2 at 99.3 eV [23]. The collected data were processed with CasaXPS software. 2.4. Loading of TAMRA on ND-NH2 The experimental reactions performed to observe the binding of physisorption and chemical binding proceeded as follows. An amount
of 100 μl of ND-NH2 (10 mg/mL in 0.1 M bicarbonate buffer, pH 8.1) was added to Eppendorf tubes along with 200 μl of 0.1 M bicarbonate buffer. Stock solutions of TAMRA and TAMRA-NHS were prepared in DMSO with concentrations of 16.8 mM and 9.89 mM, as measured by UV–Vis at the absorbance of 551 nm using an extinction coefficient of 19,000 M–1cm–1. Each series consisted of four reactions using 5, 15, 20, and 40 μl of TAMRA or 2, 10, 20, and 40 μl of TAMRA-NHS added to each ND solution. The solutions were kept at room temperature under mixing for 90 min. Next, the ND was pelleted using 6000 rpm for 5 min and the supernatant was collected. The ND pellet was then resuspended in 500 μl of 0.1 M bicarbonate buffer (pH 8.1) and centrifuged again with removal of the supernatant. The amount of dye removed from the ND was calculated from the measured absorbance units of the supernatant solutions at 551 nm. The amount of TAMRA bound to the ND was then calculated by subtracting the desorbed amount of dye from the total dye added to the reaction solution. 2.5. Field tip array fabrication The field tip arrays were fabricated on 100 mm n-type silicon wafers with a silicon nitride (300 nm) surface grown via chemical vapor deposition serving as a chemical etch mask. The nitride was patterned using a negative photoresist on a Karl-Suss MA-6 mask aligner followed by reactive ion etching. A mixture of concentrated hydrofluoric, nitric, and acetic acids (HNA) in volumetric ratios of 0.025 to 0.95 to 0.025, respectively, was used for the silicon etch. The HNA mixture results in an isotropic silicon etch with a rate of ∼ 2.5 μm/ min (±0.1 μ/m). The patterned silicon was etched until the protective nitride pattern was released (∼ 2 min.). The resulting field tips were ∼5 μm tall; the array pattern consists of rows (2 tips wide) with tips spaced 15 μm apart in a close-packed arrangement with a row periodicity of ∼ 39 μm. Optical inspection of the tips by SEM showed that roughly 10% of the tips had the desired sharpness with a 20 nm radius of curvature. 2.6. Electrophoresis For the electrophoresis experiments using ND-NH2, T-ND, and TND-B, the diluted, aqueous solution was placed in a 1 × 1 × 1 cm cell between the negative electrode (the doped Si, nanoflake, gold line device, or a field tip array) and a positive electrode. A potential of 1–10 V was applied for a period of 5–20 min. Target capture of streptavidin prior to electrophoresis was completed by the addition of FITC-streptavidin labeled to a solution of T-ND-B. Subsequent centrifugation and water washing was completed to remove some of the salt. This solution was electrophoresed for 20–45 min at 10 V on a variety of electrodes. Electrode substrates were imaged using a TRITC excitation light source (exciter HQ535/50, emitter HQ610/75 from Chroma Technology Corp.) with a magnification of 1000× using an oil immersion lens fitted onto an inverted Nikon IX-71 fluorescence microscope with a 100 W mercury lamp. The images were captured using IP Lab software and were falsely colored. After the substrates were incubated with 1 mL of a 1% FITC-streptavidin solution, the substrates were imaged using a FITC excitation light source (exciter HQ480/40, emitter HQ535/ 50 from Chroma Technology Corp) and were falsely colored. Scanning electron microscopy images of the samples were taken with a Zeiss Supra 25 at 2–3 · 10− 6 Torr vacuum using 100 k X magnification. 3. Results and discussion In this work, we collected biological targets by electrophoresis using surface functionalized nanodiamond capture probes, see Fig. 1. In this way, nanodiamonds are analogous to other bead probes which capture and collect biomolecular targets. In this work, we found that there are advantages to using nanodiamond bioprobes. For example, the capture
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Fig. 2. FTIR spectra of ND starting material I6 (a) and aminated ND (b). Spectra are taken for ND-KBr pellet exposed to air and treated in IR cuvette under vacuum conditions at 100 °C (I6 and ND-NH2) and at 170 °C (ND-NH2) to remove adsorbed water. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
of solution targets is more efficient with nanometer-sized particles since mixing is more thorough. Because of their irregularly shaped aggregates, nanodiamonds have a greater surface area to volume ratio as compared to similarly-sized spherical particles; thus, nanodiamond probes should exhibit greater capture efficiency. Also, it was observed that nanodiamonds can be effectively mixed in a solution illuminated with UV, blue, or green light. Thus, facile solution mixing is possible with remote control; this property may be advantageous in a closed system, such as a microfluidic cell, for example. Once the solution is mixed thoroughly, electrophoresis may be performed in solution with modest voltages and rapid collection times. Finally, the collected target may be identified using a fluorescently-labeled target as visualized on the electrode substrate. Thus, it is conceivable that an array of target-selective ND probes, containing proteins, antibodies, or DNA, for example, could be used for diagnostic screening purposes. The production of target-selective nanodiamond capture probes involved the direct surface conjugation of biotin and TAMRA dye. To do this, standard wet chemistry synthetic methods were used to alter the heterogeneous polydispersed I6 nanodiamond material. Initially, the fully reduced form, the ND-OH product was formed, creating a starting material with greater chemical homogeneity. This step was followed by subsequent reactions to produce the ND-NH2 product that forms a stable amide bond with TAMRA-NHS and biotin-NHS.
adsorbed water provides strong absorption bands in the 3500–3300 (max 3420 cm− 1 νOH) and 1620–1630 cm− 1 (bending mode) regions. As can be seen from Fig. 2a for the dehydrated I6 sample, adsorbed
3.1. ND-NH2 characterization Considerable effort was made to characterize our ND products. The FTIR spectra for the nanodiamond starting material I6 and the reactive product ND-NH2 are shown in Fig. 2. Due to oxidative treatments of soot, the surface of I6 ND contains oxygen-containing groups, such as: ≥C–OH (3200–3600 cm− 1 for νO–H in water, hydroxyl groups in carboxylic or tertiary alcohol), ≥C–O–C≤ (ν 1100–1370 cm− 1 for ether, acid anhydride, lactones, epoxy groups), NC = O group (1700–1865 cm −1 for ketonic, carboxylic, acid anhydrides groups, ester and lactones), see Fig. 2a [24]. The I6 sample also contains an appreciable amount of C–H groups (2800–3000 cm− 1 region). It is important to note that
Fig. 3. High resolution N1s XPS spectra of ND starting material I6 (a) and aminated ND, ND-NH2 (b). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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water related peaks are strongly diminished and leave a low intensity broad band with a peak at 3258 cm− 1 which can be attributed to hydroxyl groups in carboxylic species. The peak at 1630 cm− 1 also diminishes in the dehydrated sample but does not completely disappear even with heating at 200 °C in vacuum (spectrum not shown). This residual peak can be attributed to other species, such as amide related bands as assigned by Jiang et al. [25]. The production of target-selective nanodiamond capture probes involved the direct surface conjugation of biotin and TAMRA. To do this, ND was surface functionalized with amines (ND-NH2) that are reactive to NHS ester conjugates. Initially, using the heterogeneous polydispersed I6 nanodiamond material, the fully reduced ND-OH product was formed. This ND-OH product has greater chemical homogeneity, improving the product yield and providing more precise product characterization, a detailed analysis of FTIR spectra for all samples in the intermediate chemical reactions will be reported elsewhere. From the FTIR spectra of I6 and ND-OH (spectra not shown), the fully reduced product contains mostly O–H stretching frequencies and has lost the carbonyl containing functional groups. Also, an increase in the intensity of O–H stretching at 3450 cm− 1 as well as an increase in intensity of OH in-plane bending at 1387 cm− 1 were observed. The fully reduced product was tosylated and subsequently converted to
the ND-CN product (characteristic C ≡ N stretch at ∼2300 cm− 1, spectra not shown). Reduction of the nitrile group on ND-CN produced the final ND-NH2 product. The FTIR spectra of ND-NH2 taken in air and after dehydration at 100 °C and 170 °C in vacuum are shown in Fig. 2b. There is a dramatic difference in spectra of dehydrated I6 and dehydrated ND-NH2 samples in the 3000–3600 cm− 1 region, indicating presence of N–H groups in the ND-NH2 sample. The N–H bond has a strong absorption at 3490 cm− 1 (ν-as) and 3400 cm− 1(ν-s). In the case of inter-molecular hydrogen bonds, additional peaks appear in the region 3000–3300 cm− 1. Amines show an N–H peak between 3300–3450 cm− 1 [24]. Jiang et al. [25] assign the following NH frequencies for the NH bond within an amide group: νNH between 3360–3320 cm− 1, combination bands of δNH and νCH between 1650 and 1620 cm− 1 and 1200–1305 cm− 1. In the spectra of ND-NH2 in Fig. 2b the peak 1630 cm− 1 diminishes as the dehydration temperature is increased, suggesting that this peak is mostly related to adsorbed water. In the dehydrated sample, the peak 1959 cm− 1 also became pronounced (Fig. 2b). It can be attributed to C– NH+3 (OH) groups (similar in water solutions of ammonia) [24]. It should be also noted that after dehydration at 170 °C in vacuum (and at 220 °C, spectra not shown), water still can be adsorbed onto amino groups, forming strong Lewis acid (H in water)–Lewis base (nitrogen) bonds within a band peaking at 3412 cm− 1 according to Ji et al. This group also
Fig. 4. Schematic reaction of amine-modified nanodiamond with the NHS ester on biotin and TAMRA to create the double conjugate TAMRA-ND-Biotin (T-ND-B).
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The amination reaction results in a dramatic increase in the overall content of sp3 type nitrogen, which can be attributed to the formation of amine species on the surface of the nanodiamond (Fig. 3b). Stable ND probe suspensions that do not agglomerate are important for effectively capturing targets and for efficient collection by electrophoresis. The aqueous ND-NH2 suspension formed a colloidal suspension that was stable for at least 8 h. Zeta potential of ND-NH2 product is +40 mV (at 0.1 wt.%), in accordance with high colloidal stability, and has an average size of 140 nm. 3.2. ND conjugate probes
Fig. 5. The ND-T conjugate remains colored by TAMRA (top row), while the physisorbed samples have no visible color (bottom row) after several washings. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
suggests the existence of another form of adsorbed water on Lewis base type species, which is accompanied by the formation of hydrogen bonds between the adsorbed water molecules with a corresponding shoulder at 3240 cm− 1 [22]. In principle, the shoulder seen at 3120 cm− 1 after 100 °C dehydration in vacuum of the ND-NH2 sample, which disappears after dehydration at higher temperature (Fig. 2b), can be attributed to this type of bonding. In order to further confirm the formation of aminated ND, we performed X-ray photoelectron spectroscopy (XPS) (XPS data for all ND intermediate products will be reported elsewhere). Fig. 3 illustrates the XPS spectra for the starting I6 and aminated ND samples. After deconvolution, three characteristic peaks are revealed in the initial I6 and aminated ND-NH2 samples: 399.8, 400.8 and 403.6 eV. The peak at 399.8 eV is indicative of nitrogen in a sp3 type environment [26]. The peak at 400.8 eV is indicative of nitrogen in a sp2 type environment (imine) [27]. Gouzman et al. attributes 401.3 eV peak in N-implanted CVD diamond films to NN–Nb and –N = N– species [26]. Butenko et al. speculate that 400.7 eV peak in DND sample could belong to a slightly positively charged substitutional nitrogen atom having a three neighbor carbon atom [28]. The peak at 403.6 eV in all of the samples is possibly indicative of N–O species (nitro, nitroso) [29,30]. A similar peak at 404.0 eV was attributed to C–O–N species incorporated to the ND lattice [31]. Given that there is little amine present in the pristine sample, the sp3 nitrogen (Fig. 3a) is most likely that of a nitrogen lattice defect. Noticeable changes of the N1s line shape (appearance of an asymmetrical peak) is taking place for the sample undergone amination reaction.
A fluorescently-labeled TAMRA, biotinylated nanodiamond probe (T-ND-B) was derived from the amine-modified nanodiamond (NDNH2) and the reactive N-hydroxysuccinimidyl ester conjugate of TAMRA and biotin (Fig. 4). With this product, it is possible to detect the nanodiamond probe (T-ND-B) from the fluorescence emission of TAMRA. Thus streptavidin (labeled with FITC) can be located on the nanodiamond probe by fluorescence microscopy using TAMRA and FITC filter sets. The reaction between ND-NH2 and NHS-TAMRA was evaluated by comparing it to the amount of TAMRA that physisorbes onto the ND. After the reaction was performed with TAMRA-NHS and TAMRA without NHS, the samples were washed by centrifugation/pelleting and removal of the supernatant. The pellets for the physisorbed reaction were grey, having no observable dye, while the ND pellet for the reaction with TAMRA-NHS were pink from bound dye molecules, see Fig. 5. For an amount of 740 μM TAMRA in the reaction mixture, ten times as much TAMRA was bound to ND using TAMRA-NHS as compared to TAMRA only in the physisorbed control. The amount of TAMRA loaded onto the ND was 285 μmol/g, comprising of chemical and physical adsorption, whereas 24 μmol/g was loaded onto ND-NH2 in the physisorbed sample. Therefore, the amount of chemical loading for TAMRA on ND-NH2 is 261 μmol/g and is equivalent to a density of 0.8 molecules/nm2, using the I6 ND specific surface area of 200 m2/g as estimated by the BET (Brunauer Emmett Teller) method. By microscopy, TAMRA luminescence can be seen on the nanodiamond particles, see Fig. 6. The T-ND product remained in a colloidal solution for at least 8 h and had a similar zeta potential to ND-NH2 (+40 mV). The T-ND conjugate, however, was larger with a size of 200 nm. Similar to the starting material, sedimentation of T-ND was observed after overnight incubation at room temperature. Brief sonication was sufficient to resuspend the solid without noticeable degradation of the dye or dye–ND complex. The ND products used in this work do not require stabilizing additives, such as sodium oleate or alkaline media [32]; they form stable colloidal suspensions. In fact, it was recently demonstrated that our commercial product T-ND (www.itc-inc.
Fig. 6. Microscopy images of well dispersed T-ND particles (1000×) using bright field (left) and fluorescence emission with green excitation light (right). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 7. SEM images (100 k X) of ND-NH2 on conductive silicon after electrophoresis at the specified time and voltage in a 1 cm cell containing 1 mL ND solution in water.
org) can serve as a tracer probe for localization of NDs in neuroblastoma cells over a period of 24 h. In addition, the presence of the conjugated dye did not affect the biocompatibility of the nanodiamond [33]. 3.3. Nonspecific protein binding Nonspecific binding of NDs has been studied by several groups [4–6,17] and provides a simple means of collecting biomolecules, while centrifugation is used for separation of target analytes. On the other hand, target-specific ND probes allow for the detection of specific biological targets, while electrophoresis can be used as a collection method. However, for target-specific ND probes, it is essential to measure the extent of nonspecific binding. Nonspecific binding of ND-NH2 to the protein streptavidin was tested by adding FITC-streptavidin (FITC-S) to a solution of ND-NH2. From this solution, fluorescence emission of streptavidin was observed on the particulate matter using fluorescence and bright field microscopy. Streptavidin binding could not be disrupted using a protein blocking agent consisting of 1% BSA, 0.05% Tween 20, and 1×
PBS. Disruption of protein binding was also tested using only BSA and Tween, since PBS can cause agglomeration of NDs; disruption did not occur in this case. However, protein adsorption was disrupted by electrophoresis at 10 V/cm, as observed by the agglomeration of protein at the positive electrode and by the absence of fluorescence on the negative electrode. Ionic bonding between ND-NH2 and FITCstreptavidin is expected to dissociate during electrophoresis. Thus, in our experiments using electrophoresis of captured streptavidin, we expect to observe only specific binding. It was previously shown that nonspecific binding can be reduced on diamond films by the addition of a monolayer of ethylene glycol [34]. In analogy, the surface of nanodiamond particles was conjugated with polyethylene glycol (PEG). To do this, trimethoxysilicon-polyethylene glycol was added to the starting material ND-OH in methylene chloride and the solution was stirred at room temperature for 12 h. The product was washed multiple times in water. Fluorescence microscopy was used to image a solution of FITC-S mixed with ND-PEG. From the fluorescence and bright field images, FITC-S remained adsorbed onto the ND, however this result may be caused by low surface coverage of ND by PEG.
Fig. 8. Fluorescence image (left) and scanning electron microscopy image (right) of T-ND collected on a field tip array electrode applying 10 V/cm for 5 min.
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Fig. 9. Fluorescence microscopy images of the double conjugate T-ND-B and FITC-streptavidin imaging TAMRA (left) and FITC (right). In most locations, the equivalent emission intensity is observed. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.4. Electrophoretic capture DNDs with a high zeta potential over a wide pH range in water suspensions are able to travel over millimeters in distance in 1 min. This is rapid considering that the total collection time is on the order of a few minutes. Rapid collection of ND products by electrophoresis will promote the utility of this method as a simple means to collect target analytes that bind NDs. The zeta potential of our nanodiamond products are +40 mV and thus these particles will travel equally far using similar applied fields. Several different types of electrodes were used in this work, including conductive silicon, gold lines, lines with deposited carbon nanoflakes, and silicon field tip arrays (FTAs). Field tip arrays have been previously used to collect nanodiamonds for field emission experiments [35]. Experimental work was completed to determine the dependence of distance traveled as a function of applied electric field. Using a 0.005% solution of ND-NH2, a potential of 1 V/cm or 10 V/cm was applied for 30 s or 300 s between two conductive silicon substrates. The amount of ND particles that were collected on the negative electrode surfaces were compared from their SEM images, see Fig. 7. Saturation of NDs on the substrate is observed at 10 V for an electrophoretic duration of 30 s and 300 s, while a marked difference between these two collection times is observed for an applied field of 1 V. The rapid velocity of ND particles depletes the region closest to the electrode, thus the substrate saturates more rapidly at 10 V. It was found that the current measured in the electrophoresis cell exponentially decays as a function of time (data not shown) suggesting that the ND solution forms a dielectric film that reduces the speed of particle deposition. The speed of target-probe collection may be improved by either increasing the electric field or the ND surface charge density.
T-ND was observed on the tips of FTAs by fluorescence microscopy and by scanning electron microscopy (Fig. 8). T-ND also migrated to planar surfaces at similar potentials; the high electric field enhancement from FTAs is not required for the collection of NDs, but the specific pattern of adsorption on the FTAs aids in the visualization of the captured probe using fluorescent microscopy. 3.5. Specific protein binding The ND-NH2 product was bifunctionalized with biotin and TAMRA fluorophore (Fig. 4) using an excess of biotin to TAMRA (10:1). Extensive washing of the solid precipitate did not remove its pink color. Unbound biotin was removed using a microcon 30,000 MW filter. The fluorescence emission signal of TAMRA was observed on the particulate matter using bright field microscopy. T-ND-B formed stable aqueous suspensions with a zeta potential of +40 mV. The solution fluorescence intensity from FITC and TAMRA are localized on ND particles (Fig. 9). In some regions, the fluorescence intensity of FITC does not correspond to a similar intensity of TAMRA. This may be caused by different ratios of attachment for biotin and TAMRA on the polydispersed nanodiamond particles. The double conjugate was collected at the negative electrode onto a FTA and imaged with a fluorescence microscope. A fluorescence emission pattern from TAMRA was seen on the FTA. After the FTA was incubated in a streptavidin solution (Fig. 10), a similar pattern of intensity was observed for FITC-S emission. This result suggests that TND-B binds streptavidin. Electrophoresis of the captured streptavidin target on the T-NDBiotin probe was evaluated. After incubation of 1% FITC-S and 10% T-
Fig. 10. Fluorescence emission of TAMRA (left) after 5 min electrophoresis of T-ND-B at 10 V/cm and after incubation with FITC-S (right). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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ND-B in 1× PBS buffer, the salt was removed from the solid particulate by repeated centrifugation with pellet washing. After electrophoresis at 10 V/cm on a gold line electrode, fluorescence emission of TAMRA and FITC were observed on the active electrode area. In another experiment, T-ND-B was electrophoresed onto an electrode. The electrode was then treated with reducing agent dithiothreitol in order to break the disulfide bond in our biotin moiety; this was followed by incubation with FITC-S. Fluorescence microscopy showed little FITC-S emission in the presence of high emission intensity of TAMRA. This result suggests that streptavidin binds to biotin on the probe T-ND-B. 4. Conclusions In this work, electrophoresis was demonstrated as a means to collect nanodiamond target probes. A variety of chemically functionalized NDs were collected on different types of electrodes, including gold lines, carbon nanoflake lines, and silicon field tip arrays at modest potentials (10 V/cm) and rapid times (5 min). The nanodiamonds used in these experiments form stable colloidal solutions with high zeta potentials. These NDs were chemically functionalized with biotin for streptavidin capture and TAMRA for fluorescence detection. To achieve the highest yield and purity of products, the ND starting material was fully reduced to form the ND-OH product; this product was subsequently functionalized in several steps to form the aminated ND, ND-NH2. The aminated ND was reactive with the NHS ester of TAMRA and biotin, forming the bifunctional conjugate TAMRA-NDBiotin. This study shows that the T-ND-B conjugate can be used as a target-specific solid-phase capture probe that binds streptavidin, while the probe or probe/target complex may be collected by electrophoresis. Electrophoretic collection may be useful where collection by centrifugation is not possible, such as in microfluidic formats. Nanodiamonds are unique capture probes since they are chemically and physically robust, have a large surface area to volume ratio, fluoresce in many colors without photobleaching, and are easily modified by standard wet chemistry methods. In addition, nanodiamonds are useful for fluorescence labeling since they are transparent in the visible wavelength range, have a large index of refraction, and have irregular surface shapes that allow for a highly dense receptor binding capacity. Although photoluminescence of nanodiamonds may be enhanced using physical methods, direct conjugation of fluorescent dyes to nanodiamond surfaces provides a facile means of producing fluorescent nanodiamonds with well-defined spectral characteristics. Acknowledgments The authors are thankful for the support from the Army Research Laboratory under grant W911NF-04-2-0023 and the Defense Advanced
Research Programs Agency/Air Force Office under grant AFOSR FA955004-1-0440. References [1] O. Shenderova, D. Gruen (Eds.), Ultrananocrystalline Diamond, William-Andrew, 2006. [2] A. Kruger, Angew. Chem.-Int. 45 (2006) 6426. [3] K.B. Holt, Philosophical Transactions of the Royal Society A, 2007. [4] V.S. Bondar, I.O. Pozdnyakova, A.P. Puzyr, Phys. Solid State 46 (2004) 758. [5] X.L. Kong, L.C.L. Huang, C.M. Hsu, W.H. Chen, C.C. Han, H.C. Chang, Anal. Chem. 77 (2005) 259. [6] X.L. Kong, L.C.L. Huang, S.C.V. Liau, C.C. Han, H.C. Chang, Anal. Chem. 77 (2005) 4273. [7] A.M. Schrand, H.J. Huang, C. Carlson, J.J. Schlager, E. Osawa, S.M. Hussain, L.M. Dai, J. Phys. Chem. B 111 (2007) 2. [8] V. Grichko, V. Grishko, O. Shenderova, Nanobiotechnol 2 (2007) 37. [9] S.-J. Yu, M.-W. Kang, H.-C. Chang, K.-M. Chen, Y.-C. Yu, J. Am. Chem. Soc. 127 (2005) 17604. [10] C.C. Fu, H.Y. Lee, K. Chen, T.S. Lim, H.Y. Wu, P.K. Lin, P.K. Wei, P.H. Tsao, H.C. Chang, W. Fann, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 727. [11] O. Shenderova, V. Grichko, S. Hens, J. Walsh, Diamond Relat. Mater. 16 (2007) 2003. [12] T. Tsubota, S. Tanii, S. Ida, M. Nagata, Y. Matsumoto, Diamond Relat. Mater. 13 (2004) 1093. [13] T. Tsubota, T. Ohno, H. Yoshida, K. Kusakabe, Diamond Relat. Mater. 15 (2006) 668. [14] T. Nakamura, M. Hasegawa, K. Tsugawa, T. Ohana, M. Ishihara, Y. Koga, Diamond Relat. Mater. 15 (2006) 678. [15] Y. Liu, Z.N. Gu, J.L. Margrave, V.N. Khabashesku, Chem. Mat. 16 (2004) 3924. [16] A. Kruger, Y.J. Liang, G. Jarre, J. Stegk, J. Mater. Chem. 16 (2006) 2322. [17] L.C. Huang, H.C. Chang, Langmuir 20 (2004) 5879. [18] I. Zhitomirsky, Adv. Colloid Interface Sci. 97 (2002) 279. [19] A.M. Affoune, B.L.V. Prasad, H. Sato, T. Enoki, Langmuir 17 (2001) 547. [20] E.N. Loubnin, S.M. Pimenov, A. Blatter, F. Schwager, P.Y. Detkov, New Diam. Front. Carbon Technol. 9 (1999) 273. [21] O. Shenderova, I. Petrov, J. Walsh, V. Grichko, V. Grishko, T. Tyler, G. Cunningham, Diamond Relat. Mater. 15 (2006) 1799. [22] Shengfu Ji, Tianlai Jiang, Kang Xu, Shuben Li, Appl. Surf. Sci. 133 (1998) 231. [23] D.S. Blair, J.W. Rogers Jr., C.H.F. Peden, J. Appl. Phys. 67 (1990) 2066. [24] W. William Simons, The Sadtler Handbook of Infrared Spectra, Sadtler, Philadelphia, 1978. [25] T. Jiang, K. Xu, Carbon 33 (1995) 1663. [26] I. Gouzman, R. Brener, A. Hoffman, Surf. Sci. 333 (1995) 283. [27] M.M.T. Khan, S. Srivastava, Polyhedron 7 (1988) 1063. [28] M. Yeganeh, P.R. Coxon, A.C. Brieva, V.R. Dhanak, L. Siller, Yu V. Butenko, Phys. Rev. B 75 (2007) 155404. [29] C.D. Batich, D.S. Donald, J. Am. Chem. Soc. 106 (1984) 2758. [30] R. Nakagaki, D.C. Frost, C.A. Mcdowell, J. Electron Spectrosc. Relat. Phenom. 22 (1981) 289. [31] Yu.V. Butenko, V.L. Kuznetsov, E.A. Paukshtis, A.I. Stadnichenko, I.N. Mazov, S.I. Moseenkob, A.I. Boronin, S.V. Kosheev, Fuller., Nanotub., Carbon Nanostruct. 14 (2006) 557. [32] X. Xua, Z. Yu, Y. Zhu, B. Wang, Diamond Relat. Mater. 14 (2005) 206. [33] Amanda Michelle Schrand, Characterization and in vitro biocompatibility of engineered nanomaterials, The School of Engineering University of Dayton, Dayton, 2007, p. 276. [34] R.J. Hamers, J.E. Butler, T. Lasseter, B.M. Nichols, J.N. Russell Jr, K.-Y. Tse, W. Yang, Diamond Relat. Mater. 14 (2005) 661. [35] M. Hajra, N.N. Chubun, A.G. Chakhovskoi, C.E. Hunt, K. Liu, A. Murali, S.H. Risbud, T. Tyler, V. Zhirnov, J. Vac. Sci. Technol. B 21 (2003) 458.
Contents lists available at ScienceDirect
Diamond & Related Materials j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / d i a m o n d
Nanodiamond bioconjugate probes and their collection by electrophoresis Suzanne Ciftan Hens a,⁎, Garry Cunningham a, Talmage Tyler a, Sergey Moseenkov b, Vladimir Kuznetsov b, Olga Shenderova a a b
International Technology Center, 8100 Brownleigh Drive, Suite 120, Raleigh, NC 27617, United States Boreskov Institute of Catalysis SB RAS, Lavrentiev Ave. 5, Novosibirsk, 630090, Russia
A R T I C L E
I N F O
Article history: Received 29 June 2007 Received in revised form 25 February 2008 Accepted 14 March 2008 Available online 1 April 2008 Keywords Diamond Nanotechnology Surface Chemistry Bioconjugation Electrophoresis Streptavidin
A B S T R A C T The application of detonation nanodiamonds (NDs) as probes for protein capture and electrophoretic collection was investigated. NDs were chemically modified in a series of reactions to produce a ND-NH2 product that had increased chemical homogeneity. The product was characterized by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). FTIR spectra were taken using an IR vacuum cuvette and the samples were dehydrated at different temperatures. The ND-NH2 product was capable of conjugating to N-hydroxysuccinimide derivatives of TAMRA and biotin. We calculated that the number of chemically attached TAMRA molecules on ND-NH2 was ∼ 1 molecule/nm2. The singly conjugated TAMRA-ND (T-ND) and doubly conjugated TAMRA-ND-Biotin (T-ND-B) products formed stable aqueous colloidal suspensions. T-ND and T-ND-B were collected on planar electrodes and silicon field tip arrays using a field of 10 V/cm. The rate of collection for the aminated ND is dependent upon field strength and an exponential decrease in current was observed as a function of time. Streptavidin was captured by the T-ND-B bioconjugate probe and this nanoparticle–protein complex was collected from solution by electrophoresis. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Detonation nanodiamond (DND) particles [1] are an inexpensive complementary material to CVD diamond films and have exceptional properties that are useful for a diverse set of applications, including biomolecular scaffolds, fluorescent probes, and gene delivery vehicles [2,3]. In terms of biomolecular scaffolds, nanodiamonds may physically adsorb proteins and DNA oligos for biomolecular separation and preconcentration [4–6]. As fluorescent probes, NDs may be used as cellular tracer probes due to their low cytotoxicity [7] and their fluorescent properties. And since nanodiamonds are physically strong, DNDs, physisorbed with plasmid DNA, have been used as ballistic delivery vehicles for gene transfection into bacteria [8]. Nanodiamonds fluoresce with different colors, have little photobleaching and blinking characteristics, and are highly luminescent after high energy bombardment that increases their number of nitrogen-vacancy centers [9,10]. Unlike many nanoparticles used as biomolecular probes, the optical transparency of nanodiamonds in the visible wavelength range [11] does not inhibit luminescence of bound fluorophores. In addition, nanodiamonds are compatible as microbiological fluorescent probes since they can be sterilized with high temperature and pressure. ⁎ Corresponding author. Tel.: +1 919 881 0500. E-mail address: [email protected] (S.C. Hens). URL: http://www.itc-inc.org (S.C. Hens). 0925-9635/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.diamond.2008.03.020
Even though the nanodiamond core is physically and chemically strong, its rich surface chemistry is easily modified with a variety of methods. Surface functionalization on diamond includes radical based reactions, producing carboxylic acid, NO2 surface groups [12,13], and fluorine surface groups [14]. Standard wet chemical methods may be used, including the use of fluorinated nanodiamond particles that can be converted to alkyl, amino, and amino acid derivatives [15]. In terms of bioconjugation reactions, nanodiamonds have been derivatized through their direct linkage to reactive surface functional groups to make ND conjugated peptides [16]. And direct conjugation of fluorescent dye and protein has been possible using the free amines from poly-lysine coated nanodiamonds [17]. Nanodiamonds have been used for electrophoretic applications due to their high electrophoretic mobility and controlled surface charges. In fact, electrophoresis of DNDs is a well established area and is used to deposit DNDs as a seed material for CVD growth of diamond films [18,19], and it is used for the co-deposition of a variety of metals during electroplating [20]. While centrifugation is commonly used to collect ND-bound targets from solution, we have found that electrophoresis may also be used for this purpose. Electrophoretic collection of targetbound NDs is practical since the velocity of a ND particle with a high zeta potential of +40 mV will travel a distance of ∼ 2 mm in a short period of time (1 min) using an applied field of only 10 V/cm, as calculated from the particle's electrophoretic mobility [18]. In this work, nanodiamonds are used as target-specific capture probes in order to bind and collect their target, streptavidin, using
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Fig. 1. A schematic representation of the collection of target analytes using nanodiamonds. The T-ND-B nanodiamond probe has biotinylated (B) and TAMRA (T) functionalized to its surface for the capture of the target analyte FITC-streptavidin (S-F) (Step 1). The ND probe mixes and captures streptavidin. Green excitation light may be used to mix and visualize TAMRA emission (Step 2). Under electrophoresis, the captured analyte can be collected from suspension at modest voltages with many types of electrodes, including the field tip array electrode (shown) (Step 3). The captured and collected analyte on the nanodiamond probe may be detected using two excitation wavelengths (Step 4). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
electrophoresis at low electric field strengths. The use of ND in this way is depicted in Fig. 1, where the ND probe is mixed in solution to bind its target and is then collected by electrophoresis for detection by fluorescence emission. To demonstrate this approach, a biotinylated nanodiamond conjugate was synthesized for the specific capture of streptavidin protein. On the same ND surface, a fluorescent dye derivative of rhodamine (carboxytetramethylrhodamine or TAMRA) was attached for detection by fluorescence microscopy. 2. Experimental All chemicals were used without modification. 5-(and-6)carboxytetramethylrhodamine succinimidyl ester (TAMRA-SE, TAMRANHS) and fluorescein isothiocyanate (FITC)-streptavidin were purchased from Invitrogen (South San Francisco, CA). Dry dimethylformamide (DMF) and Sulfo-NHS-SS-Biotin were purchased from Pierce (Rockford, IL). Unlabeled streptavidin, BSA, dimethylsulfoxide (DMSO), dithiolthreitol, potassium carbonate, carbonate buffer, and Tween 20 were purchased from Sigma Aldrich. 2.1. ND-NH2 synthesis The polydispersed nanodiamond material was produced using trinitrotoluene (TNT) and cyclotrimethylenetrinitramine (RDX) explosives by detonation in an ice-cooled chamber. The nanodiamonds have a primary particle size of 4 nm. Ch-St DND and I6 were supplied by New Technologies, Co., Chelyabinsk, Russian Federation. Ch-St was purified using a solution of chromic anhydride in sulfuric acid. The I6 samples were purified from Ch-St by ion-exchange, annealing in air, and fractionation [21]. The ND-NH2 material was produced from polydispersed I6 material containing particle sizes of 20–800 nm with average agglomerate size of 160 nm in a water suspension. Below we provide further details on the ND intermediate products for the synthesis of aminated ND (ND-NH2).
ND-OH(1): An amount of 5 g nanodiamond (I6) was added to a round bottom flask, which was then purged several times using standard Schlenk techniques. To the round bottom flask, 20 mL of degassed anhydrous tetrahydrofuran (THF) and 50 mL of a 2.0 M solution of LiAlH4 in THF were added by cannula. The sample was stirred under a nitrogen atmosphere at room temperature overnight. The reaction was quenched by dropwise addition of 1 M HCl, which solubilized the lithium and aluminum, and then the solution was basified to neutral pH. The product was collected by centrifugation and rinsed several times with water followed by acetone. The product was dried in vacuo at 100 °C for 12 h. X-ray photoelectron spectroscopy (XPS) analysis did not reveal residual lithium or aluminum. ND-OTS(2): To the solid 2 g ND-OH(1) in a round bottom flask an amount of 30 mL of anhydrous pyridine was added by syringe. The solution mixture was sonicated for 5 min. An amount of 1 g of p-toluenesulfonyl chloride or tosyl chloride (OTS) was added and the mixture was stirred at room temperature overnight. The reaction product was collected by centrifugation, rinsed several times with acetone and was dried in vacuo at 100 °C for 12 h. ND-CN(3): 1 g of ND-OTS(2) was placed in round bottom flask with 50 mL of dimethylsulfoxide (DMSO) and sonicated for 10 min. Then 1 g of potassium cyanide was added and the reaction was allowed to stir at 95 °C overnight. The product was collected by centrifugation and rinsed several times with water followed by acetone. The product was dried in vacuo at 100 °C for 12 h. ND-NH2(4): An amount of 500 mg of ND-CN(3) was placed in a round bottom flask, after which the flask was purged several times using standard Schlenk techniques. To the flask, 20 mL of a 2 M solution of LiAlH4 in THF was added and the reaction was allowed to stir at room temperature under a nitrogen atmosphere overnight. The reaction was quenched by dropwise addition of distilled water until hydrogen evolution ceased. The solution then was acidified with 1 M HCl to solubilize lithium and aluminum. The product was collected by centrifugation and washed several times with 2 M NaOH to remove Al
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(OH)3. The product was rinsed with distilled water until the runoff had a neutral pH. The product was dried in vacuo at 100 °C for 12 h. XPS analysis did not reveal residual lithium or aluminum. Zeta potential and particle sizes were measured with a Malvern Zetasizer Nano ZS instrument. The samples were prepared by addition of water to the ND solid, followed by 2 min sonication using a horntype sonicator at 35% power. Samples were measured using Malvern's plastic cuvette. 2.2. ND conjugation reactions The TAMRA-ND and TAMRA-ND-Biotin products were produced by labeling ND-NH2 with their respective succinimidyl ester conjugates (NHS-TAMRA, NHS-Biotin). For the TAMRA-ND-Biotin bioconjugate (T-ND-B), an amount of 30 mg ND-NH2 was added to 5 mL dry DMF and sonicated at 35% power for 4 min before 70 mg K2CO3 was added (pH 8). This was followed by dropwise addition of a solution of 17 mg of sulfo-NHS-SS-Biotin and 1 mg NHS-TAMRA in 1 mL dry DMF. The resulting solution was stirred at room temperature for 90 min and the solid product was collected by centrifugation at 7000 rpm, followed by extensive washing with distilled water. The final concentration of T-ND-B was 30 mg/mL. The same reaction conditions were used to produce the TAMRA-ND product. For the T-ND product, the pellet was colored pink after collection by centrifugation. The presence of an amide bond between TAMRA and ND was tested by hydrolysis. The amine bond was hydrolyzed by the addition of HCl, producing a solution of pH of 1. The solution was centrifuged at 14,000 rpm for 10 min, producing a grey colored pellet and a pink colored supernatant. 2.3. ND surface group characterization It is known, that peaks attributed to OH stretching and bending vibrations of nanodiamond adsorbed water obscure the signatures of other moieties, including NH vibrations that are important for the identification of aminated ND in the present study [22]. Thus in addition to the spectra taken in the air environment, the starting material I6 and the aminated ND samples were analyzed using a vacuum IR cuvette to avoid strong absorbance signals of adsorbed water. Samples for the analysis were prepared by homogenizing of DND in spectral grade KBr in a grinding mill and then pressed into pellets. A fixed quantity of ND sample (25 mg for all samples, except 5 mg of ND-NH2 in air due to its strong absorbance signals) was mixed with KBr powder (800 mg) and pressed with a pressure of up to 150 kg/cm2 into plates 0.5–0.7 mm thick. Tablets were placed in an IR vacuum cell and heated at different temperatures within the range 100–220 °C under vacuum (1 × 10− 2 Torr) for 2–3 h inside an IR cuvette in order to remove traces of water. After this procedure, FTIR spectra were recorded without exposure of the sample to air in order to avoid any influence of atmospheric water. Reference spectra for the empty IR vacuum cuvette (made from KBr plates) were also taken in order to register possible contribution to the sample spectra from the water and CO2 (2370 cm− 1) adsorbed on the external cuvette walls. FTIR analysis was performed using a Shimadzu FTIR-8300 spectrometer. XPS analysis was performed using a Kratos Axis Ultra with a monochromatic Al Kα X-ray source and a silicon wafer as a supporting substrate. The survey and high resolution scans were performed at 160 eV pass energy with 1 eV step and 10 eV pass energy with 0.1 eV step, respectively. The peak position calibration was done against Si 2p3/2 at 99.3 eV [23]. The collected data were processed with CasaXPS software. 2.4. Loading of TAMRA on ND-NH2 The experimental reactions performed to observe the binding of physisorption and chemical binding proceeded as follows. An amount
of 100 μl of ND-NH2 (10 mg/mL in 0.1 M bicarbonate buffer, pH 8.1) was added to Eppendorf tubes along with 200 μl of 0.1 M bicarbonate buffer. Stock solutions of TAMRA and TAMRA-NHS were prepared in DMSO with concentrations of 16.8 mM and 9.89 mM, as measured by UV–Vis at the absorbance of 551 nm using an extinction coefficient of 19,000 M–1cm–1. Each series consisted of four reactions using 5, 15, 20, and 40 μl of TAMRA or 2, 10, 20, and 40 μl of TAMRA-NHS added to each ND solution. The solutions were kept at room temperature under mixing for 90 min. Next, the ND was pelleted using 6000 rpm for 5 min and the supernatant was collected. The ND pellet was then resuspended in 500 μl of 0.1 M bicarbonate buffer (pH 8.1) and centrifuged again with removal of the supernatant. The amount of dye removed from the ND was calculated from the measured absorbance units of the supernatant solutions at 551 nm. The amount of TAMRA bound to the ND was then calculated by subtracting the desorbed amount of dye from the total dye added to the reaction solution. 2.5. Field tip array fabrication The field tip arrays were fabricated on 100 mm n-type silicon wafers with a silicon nitride (300 nm) surface grown via chemical vapor deposition serving as a chemical etch mask. The nitride was patterned using a negative photoresist on a Karl-Suss MA-6 mask aligner followed by reactive ion etching. A mixture of concentrated hydrofluoric, nitric, and acetic acids (HNA) in volumetric ratios of 0.025 to 0.95 to 0.025, respectively, was used for the silicon etch. The HNA mixture results in an isotropic silicon etch with a rate of ∼ 2.5 μm/ min (±0.1 μ/m). The patterned silicon was etched until the protective nitride pattern was released (∼ 2 min.). The resulting field tips were ∼5 μm tall; the array pattern consists of rows (2 tips wide) with tips spaced 15 μm apart in a close-packed arrangement with a row periodicity of ∼ 39 μm. Optical inspection of the tips by SEM showed that roughly 10% of the tips had the desired sharpness with a 20 nm radius of curvature. 2.6. Electrophoresis For the electrophoresis experiments using ND-NH2, T-ND, and TND-B, the diluted, aqueous solution was placed in a 1 × 1 × 1 cm cell between the negative electrode (the doped Si, nanoflake, gold line device, or a field tip array) and a positive electrode. A potential of 1–10 V was applied for a period of 5–20 min. Target capture of streptavidin prior to electrophoresis was completed by the addition of FITC-streptavidin labeled to a solution of T-ND-B. Subsequent centrifugation and water washing was completed to remove some of the salt. This solution was electrophoresed for 20–45 min at 10 V on a variety of electrodes. Electrode substrates were imaged using a TRITC excitation light source (exciter HQ535/50, emitter HQ610/75 from Chroma Technology Corp.) with a magnification of 1000× using an oil immersion lens fitted onto an inverted Nikon IX-71 fluorescence microscope with a 100 W mercury lamp. The images were captured using IP Lab software and were falsely colored. After the substrates were incubated with 1 mL of a 1% FITC-streptavidin solution, the substrates were imaged using a FITC excitation light source (exciter HQ480/40, emitter HQ535/ 50 from Chroma Technology Corp) and were falsely colored. Scanning electron microscopy images of the samples were taken with a Zeiss Supra 25 at 2–3 · 10− 6 Torr vacuum using 100 k X magnification. 3. Results and discussion In this work, we collected biological targets by electrophoresis using surface functionalized nanodiamond capture probes, see Fig. 1. In this way, nanodiamonds are analogous to other bead probes which capture and collect biomolecular targets. In this work, we found that there are advantages to using nanodiamond bioprobes. For example, the capture
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Fig. 2. FTIR spectra of ND starting material I6 (a) and aminated ND (b). Spectra are taken for ND-KBr pellet exposed to air and treated in IR cuvette under vacuum conditions at 100 °C (I6 and ND-NH2) and at 170 °C (ND-NH2) to remove adsorbed water. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
of solution targets is more efficient with nanometer-sized particles since mixing is more thorough. Because of their irregularly shaped aggregates, nanodiamonds have a greater surface area to volume ratio as compared to similarly-sized spherical particles; thus, nanodiamond probes should exhibit greater capture efficiency. Also, it was observed that nanodiamonds can be effectively mixed in a solution illuminated with UV, blue, or green light. Thus, facile solution mixing is possible with remote control; this property may be advantageous in a closed system, such as a microfluidic cell, for example. Once the solution is mixed thoroughly, electrophoresis may be performed in solution with modest voltages and rapid collection times. Finally, the collected target may be identified using a fluorescently-labeled target as visualized on the electrode substrate. Thus, it is conceivable that an array of target-selective ND probes, containing proteins, antibodies, or DNA, for example, could be used for diagnostic screening purposes. The production of target-selective nanodiamond capture probes involved the direct surface conjugation of biotin and TAMRA dye. To do this, standard wet chemistry synthetic methods were used to alter the heterogeneous polydispersed I6 nanodiamond material. Initially, the fully reduced form, the ND-OH product was formed, creating a starting material with greater chemical homogeneity. This step was followed by subsequent reactions to produce the ND-NH2 product that forms a stable amide bond with TAMRA-NHS and biotin-NHS.
adsorbed water provides strong absorption bands in the 3500–3300 (max 3420 cm− 1 νOH) and 1620–1630 cm− 1 (bending mode) regions. As can be seen from Fig. 2a for the dehydrated I6 sample, adsorbed
3.1. ND-NH2 characterization Considerable effort was made to characterize our ND products. The FTIR spectra for the nanodiamond starting material I6 and the reactive product ND-NH2 are shown in Fig. 2. Due to oxidative treatments of soot, the surface of I6 ND contains oxygen-containing groups, such as: ≥C–OH (3200–3600 cm− 1 for νO–H in water, hydroxyl groups in carboxylic or tertiary alcohol), ≥C–O–C≤ (ν 1100–1370 cm− 1 for ether, acid anhydride, lactones, epoxy groups), NC = O group (1700–1865 cm −1 for ketonic, carboxylic, acid anhydrides groups, ester and lactones), see Fig. 2a [24]. The I6 sample also contains an appreciable amount of C–H groups (2800–3000 cm− 1 region). It is important to note that
Fig. 3. High resolution N1s XPS spectra of ND starting material I6 (a) and aminated ND, ND-NH2 (b). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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water related peaks are strongly diminished and leave a low intensity broad band with a peak at 3258 cm− 1 which can be attributed to hydroxyl groups in carboxylic species. The peak at 1630 cm− 1 also diminishes in the dehydrated sample but does not completely disappear even with heating at 200 °C in vacuum (spectrum not shown). This residual peak can be attributed to other species, such as amide related bands as assigned by Jiang et al. [25]. The production of target-selective nanodiamond capture probes involved the direct surface conjugation of biotin and TAMRA. To do this, ND was surface functionalized with amines (ND-NH2) that are reactive to NHS ester conjugates. Initially, using the heterogeneous polydispersed I6 nanodiamond material, the fully reduced ND-OH product was formed. This ND-OH product has greater chemical homogeneity, improving the product yield and providing more precise product characterization, a detailed analysis of FTIR spectra for all samples in the intermediate chemical reactions will be reported elsewhere. From the FTIR spectra of I6 and ND-OH (spectra not shown), the fully reduced product contains mostly O–H stretching frequencies and has lost the carbonyl containing functional groups. Also, an increase in the intensity of O–H stretching at 3450 cm− 1 as well as an increase in intensity of OH in-plane bending at 1387 cm− 1 were observed. The fully reduced product was tosylated and subsequently converted to
the ND-CN product (characteristic C ≡ N stretch at ∼2300 cm− 1, spectra not shown). Reduction of the nitrile group on ND-CN produced the final ND-NH2 product. The FTIR spectra of ND-NH2 taken in air and after dehydration at 100 °C and 170 °C in vacuum are shown in Fig. 2b. There is a dramatic difference in spectra of dehydrated I6 and dehydrated ND-NH2 samples in the 3000–3600 cm− 1 region, indicating presence of N–H groups in the ND-NH2 sample. The N–H bond has a strong absorption at 3490 cm− 1 (ν-as) and 3400 cm− 1(ν-s). In the case of inter-molecular hydrogen bonds, additional peaks appear in the region 3000–3300 cm− 1. Amines show an N–H peak between 3300–3450 cm− 1 [24]. Jiang et al. [25] assign the following NH frequencies for the NH bond within an amide group: νNH between 3360–3320 cm− 1, combination bands of δNH and νCH between 1650 and 1620 cm− 1 and 1200–1305 cm− 1. In the spectra of ND-NH2 in Fig. 2b the peak 1630 cm− 1 diminishes as the dehydration temperature is increased, suggesting that this peak is mostly related to adsorbed water. In the dehydrated sample, the peak 1959 cm− 1 also became pronounced (Fig. 2b). It can be attributed to C– NH+3 (OH) groups (similar in water solutions of ammonia) [24]. It should be also noted that after dehydration at 170 °C in vacuum (and at 220 °C, spectra not shown), water still can be adsorbed onto amino groups, forming strong Lewis acid (H in water)–Lewis base (nitrogen) bonds within a band peaking at 3412 cm− 1 according to Ji et al. This group also
Fig. 4. Schematic reaction of amine-modified nanodiamond with the NHS ester on biotin and TAMRA to create the double conjugate TAMRA-ND-Biotin (T-ND-B).
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The amination reaction results in a dramatic increase in the overall content of sp3 type nitrogen, which can be attributed to the formation of amine species on the surface of the nanodiamond (Fig. 3b). Stable ND probe suspensions that do not agglomerate are important for effectively capturing targets and for efficient collection by electrophoresis. The aqueous ND-NH2 suspension formed a colloidal suspension that was stable for at least 8 h. Zeta potential of ND-NH2 product is +40 mV (at 0.1 wt.%), in accordance with high colloidal stability, and has an average size of 140 nm. 3.2. ND conjugate probes
Fig. 5. The ND-T conjugate remains colored by TAMRA (top row), while the physisorbed samples have no visible color (bottom row) after several washings. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
suggests the existence of another form of adsorbed water on Lewis base type species, which is accompanied by the formation of hydrogen bonds between the adsorbed water molecules with a corresponding shoulder at 3240 cm− 1 [22]. In principle, the shoulder seen at 3120 cm− 1 after 100 °C dehydration in vacuum of the ND-NH2 sample, which disappears after dehydration at higher temperature (Fig. 2b), can be attributed to this type of bonding. In order to further confirm the formation of aminated ND, we performed X-ray photoelectron spectroscopy (XPS) (XPS data for all ND intermediate products will be reported elsewhere). Fig. 3 illustrates the XPS spectra for the starting I6 and aminated ND samples. After deconvolution, three characteristic peaks are revealed in the initial I6 and aminated ND-NH2 samples: 399.8, 400.8 and 403.6 eV. The peak at 399.8 eV is indicative of nitrogen in a sp3 type environment [26]. The peak at 400.8 eV is indicative of nitrogen in a sp2 type environment (imine) [27]. Gouzman et al. attributes 401.3 eV peak in N-implanted CVD diamond films to NN–Nb and –N = N– species [26]. Butenko et al. speculate that 400.7 eV peak in DND sample could belong to a slightly positively charged substitutional nitrogen atom having a three neighbor carbon atom [28]. The peak at 403.6 eV in all of the samples is possibly indicative of N–O species (nitro, nitroso) [29,30]. A similar peak at 404.0 eV was attributed to C–O–N species incorporated to the ND lattice [31]. Given that there is little amine present in the pristine sample, the sp3 nitrogen (Fig. 3a) is most likely that of a nitrogen lattice defect. Noticeable changes of the N1s line shape (appearance of an asymmetrical peak) is taking place for the sample undergone amination reaction.
A fluorescently-labeled TAMRA, biotinylated nanodiamond probe (T-ND-B) was derived from the amine-modified nanodiamond (NDNH2) and the reactive N-hydroxysuccinimidyl ester conjugate of TAMRA and biotin (Fig. 4). With this product, it is possible to detect the nanodiamond probe (T-ND-B) from the fluorescence emission of TAMRA. Thus streptavidin (labeled with FITC) can be located on the nanodiamond probe by fluorescence microscopy using TAMRA and FITC filter sets. The reaction between ND-NH2 and NHS-TAMRA was evaluated by comparing it to the amount of TAMRA that physisorbes onto the ND. After the reaction was performed with TAMRA-NHS and TAMRA without NHS, the samples were washed by centrifugation/pelleting and removal of the supernatant. The pellets for the physisorbed reaction were grey, having no observable dye, while the ND pellet for the reaction with TAMRA-NHS were pink from bound dye molecules, see Fig. 5. For an amount of 740 μM TAMRA in the reaction mixture, ten times as much TAMRA was bound to ND using TAMRA-NHS as compared to TAMRA only in the physisorbed control. The amount of TAMRA loaded onto the ND was 285 μmol/g, comprising of chemical and physical adsorption, whereas 24 μmol/g was loaded onto ND-NH2 in the physisorbed sample. Therefore, the amount of chemical loading for TAMRA on ND-NH2 is 261 μmol/g and is equivalent to a density of 0.8 molecules/nm2, using the I6 ND specific surface area of 200 m2/g as estimated by the BET (Brunauer Emmett Teller) method. By microscopy, TAMRA luminescence can be seen on the nanodiamond particles, see Fig. 6. The T-ND product remained in a colloidal solution for at least 8 h and had a similar zeta potential to ND-NH2 (+40 mV). The T-ND conjugate, however, was larger with a size of 200 nm. Similar to the starting material, sedimentation of T-ND was observed after overnight incubation at room temperature. Brief sonication was sufficient to resuspend the solid without noticeable degradation of the dye or dye–ND complex. The ND products used in this work do not require stabilizing additives, such as sodium oleate or alkaline media [32]; they form stable colloidal suspensions. In fact, it was recently demonstrated that our commercial product T-ND (www.itc-inc.
Fig. 6. Microscopy images of well dispersed T-ND particles (1000×) using bright field (left) and fluorescence emission with green excitation light (right). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 7. SEM images (100 k X) of ND-NH2 on conductive silicon after electrophoresis at the specified time and voltage in a 1 cm cell containing 1 mL ND solution in water.
org) can serve as a tracer probe for localization of NDs in neuroblastoma cells over a period of 24 h. In addition, the presence of the conjugated dye did not affect the biocompatibility of the nanodiamond [33]. 3.3. Nonspecific protein binding Nonspecific binding of NDs has been studied by several groups [4–6,17] and provides a simple means of collecting biomolecules, while centrifugation is used for separation of target analytes. On the other hand, target-specific ND probes allow for the detection of specific biological targets, while electrophoresis can be used as a collection method. However, for target-specific ND probes, it is essential to measure the extent of nonspecific binding. Nonspecific binding of ND-NH2 to the protein streptavidin was tested by adding FITC-streptavidin (FITC-S) to a solution of ND-NH2. From this solution, fluorescence emission of streptavidin was observed on the particulate matter using fluorescence and bright field microscopy. Streptavidin binding could not be disrupted using a protein blocking agent consisting of 1% BSA, 0.05% Tween 20, and 1×
PBS. Disruption of protein binding was also tested using only BSA and Tween, since PBS can cause agglomeration of NDs; disruption did not occur in this case. However, protein adsorption was disrupted by electrophoresis at 10 V/cm, as observed by the agglomeration of protein at the positive electrode and by the absence of fluorescence on the negative electrode. Ionic bonding between ND-NH2 and FITCstreptavidin is expected to dissociate during electrophoresis. Thus, in our experiments using electrophoresis of captured streptavidin, we expect to observe only specific binding. It was previously shown that nonspecific binding can be reduced on diamond films by the addition of a monolayer of ethylene glycol [34]. In analogy, the surface of nanodiamond particles was conjugated with polyethylene glycol (PEG). To do this, trimethoxysilicon-polyethylene glycol was added to the starting material ND-OH in methylene chloride and the solution was stirred at room temperature for 12 h. The product was washed multiple times in water. Fluorescence microscopy was used to image a solution of FITC-S mixed with ND-PEG. From the fluorescence and bright field images, FITC-S remained adsorbed onto the ND, however this result may be caused by low surface coverage of ND by PEG.
Fig. 8. Fluorescence image (left) and scanning electron microscopy image (right) of T-ND collected on a field tip array electrode applying 10 V/cm for 5 min.
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Fig. 9. Fluorescence microscopy images of the double conjugate T-ND-B and FITC-streptavidin imaging TAMRA (left) and FITC (right). In most locations, the equivalent emission intensity is observed. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.4. Electrophoretic capture DNDs with a high zeta potential over a wide pH range in water suspensions are able to travel over millimeters in distance in 1 min. This is rapid considering that the total collection time is on the order of a few minutes. Rapid collection of ND products by electrophoresis will promote the utility of this method as a simple means to collect target analytes that bind NDs. The zeta potential of our nanodiamond products are +40 mV and thus these particles will travel equally far using similar applied fields. Several different types of electrodes were used in this work, including conductive silicon, gold lines, lines with deposited carbon nanoflakes, and silicon field tip arrays (FTAs). Field tip arrays have been previously used to collect nanodiamonds for field emission experiments [35]. Experimental work was completed to determine the dependence of distance traveled as a function of applied electric field. Using a 0.005% solution of ND-NH2, a potential of 1 V/cm or 10 V/cm was applied for 30 s or 300 s between two conductive silicon substrates. The amount of ND particles that were collected on the negative electrode surfaces were compared from their SEM images, see Fig. 7. Saturation of NDs on the substrate is observed at 10 V for an electrophoretic duration of 30 s and 300 s, while a marked difference between these two collection times is observed for an applied field of 1 V. The rapid velocity of ND particles depletes the region closest to the electrode, thus the substrate saturates more rapidly at 10 V. It was found that the current measured in the electrophoresis cell exponentially decays as a function of time (data not shown) suggesting that the ND solution forms a dielectric film that reduces the speed of particle deposition. The speed of target-probe collection may be improved by either increasing the electric field or the ND surface charge density.
T-ND was observed on the tips of FTAs by fluorescence microscopy and by scanning electron microscopy (Fig. 8). T-ND also migrated to planar surfaces at similar potentials; the high electric field enhancement from FTAs is not required for the collection of NDs, but the specific pattern of adsorption on the FTAs aids in the visualization of the captured probe using fluorescent microscopy. 3.5. Specific protein binding The ND-NH2 product was bifunctionalized with biotin and TAMRA fluorophore (Fig. 4) using an excess of biotin to TAMRA (10:1). Extensive washing of the solid precipitate did not remove its pink color. Unbound biotin was removed using a microcon 30,000 MW filter. The fluorescence emission signal of TAMRA was observed on the particulate matter using bright field microscopy. T-ND-B formed stable aqueous suspensions with a zeta potential of +40 mV. The solution fluorescence intensity from FITC and TAMRA are localized on ND particles (Fig. 9). In some regions, the fluorescence intensity of FITC does not correspond to a similar intensity of TAMRA. This may be caused by different ratios of attachment for biotin and TAMRA on the polydispersed nanodiamond particles. The double conjugate was collected at the negative electrode onto a FTA and imaged with a fluorescence microscope. A fluorescence emission pattern from TAMRA was seen on the FTA. After the FTA was incubated in a streptavidin solution (Fig. 10), a similar pattern of intensity was observed for FITC-S emission. This result suggests that TND-B binds streptavidin. Electrophoresis of the captured streptavidin target on the T-NDBiotin probe was evaluated. After incubation of 1% FITC-S and 10% T-
Fig. 10. Fluorescence emission of TAMRA (left) after 5 min electrophoresis of T-ND-B at 10 V/cm and after incubation with FITC-S (right). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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ND-B in 1× PBS buffer, the salt was removed from the solid particulate by repeated centrifugation with pellet washing. After electrophoresis at 10 V/cm on a gold line electrode, fluorescence emission of TAMRA and FITC were observed on the active electrode area. In another experiment, T-ND-B was electrophoresed onto an electrode. The electrode was then treated with reducing agent dithiothreitol in order to break the disulfide bond in our biotin moiety; this was followed by incubation with FITC-S. Fluorescence microscopy showed little FITC-S emission in the presence of high emission intensity of TAMRA. This result suggests that streptavidin binds to biotin on the probe T-ND-B. 4. Conclusions In this work, electrophoresis was demonstrated as a means to collect nanodiamond target probes. A variety of chemically functionalized NDs were collected on different types of electrodes, including gold lines, carbon nanoflake lines, and silicon field tip arrays at modest potentials (10 V/cm) and rapid times (5 min). The nanodiamonds used in these experiments form stable colloidal solutions with high zeta potentials. These NDs were chemically functionalized with biotin for streptavidin capture and TAMRA for fluorescence detection. To achieve the highest yield and purity of products, the ND starting material was fully reduced to form the ND-OH product; this product was subsequently functionalized in several steps to form the aminated ND, ND-NH2. The aminated ND was reactive with the NHS ester of TAMRA and biotin, forming the bifunctional conjugate TAMRA-NDBiotin. This study shows that the T-ND-B conjugate can be used as a target-specific solid-phase capture probe that binds streptavidin, while the probe or probe/target complex may be collected by electrophoresis. Electrophoretic collection may be useful where collection by centrifugation is not possible, such as in microfluidic formats. Nanodiamonds are unique capture probes since they are chemically and physically robust, have a large surface area to volume ratio, fluoresce in many colors without photobleaching, and are easily modified by standard wet chemistry methods. In addition, nanodiamonds are useful for fluorescence labeling since they are transparent in the visible wavelength range, have a large index of refraction, and have irregular surface shapes that allow for a highly dense receptor binding capacity. Although photoluminescence of nanodiamonds may be enhanced using physical methods, direct conjugation of fluorescent dyes to nanodiamond surfaces provides a facile means of producing fluorescent nanodiamonds with well-defined spectral characteristics. Acknowledgments The authors are thankful for the support from the Army Research Laboratory under grant W911NF-04-2-0023 and the Defense Advanced
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